Cell polarity is fundamental to the biology of most cells and is characterized by the asymmetric distribution of factors at the cell cortex (the region just beneath the plasma membrane) and in the cytoplasm. A conserved set of proteins, the PAR (PARtitioning defective) proteins regulate polarity in most polarized animal cells. While we know a great deal about how they generate cortical asymmetries, little is known about how cortical PAR proteins segregate diffusive proteins in the cytoplasm. A subset of the PAR proteins, including PAR-1 kinase, localize to two complementary domains at the cell cortex from which they orchestrate cellular polarities. We previously characterized a cytoplasmic function for PAR-1 that we propose provides a critical link between cortical and cytoplasmic polarities in the C. elegans zygote. PAR-1 localizes to a cytoplasmic concentration gradient that patterns an opposing gradient in the essential protein MEX-5 through local regulation of MEX-5's diffusion rate. In response, the RNA- binding proteins PIE-1 and POS-1 concentrate in the cytoplasm opposite MEX-5. We will combine quantitative live imaging, molecular genetic and mathematical modeling approaches to characterize the mechanisms that underlie these cytoplasmic asymmetries. The specific aims of this project are to: 1) Determine the mechanisms that establish the cytoplasmic PAR-1 gradient, 2) Determine how PAR-1 phosphorylation regulates the diffusion rate of MEX-5 and 3) Determine the mechanisms that partition PIE-1 and POS-1. Since the core polarity regulators are conserved in animals, knowledge of the principles and mechanisms that establish asymmetries in the C. elegans zygote will provide a foundation for understanding how asymmetries are generated in human cells.